PROJECT SUMMARY Analysis of structure and dynamics of membrane proteins is extremely challenging due to presence of the phase-separation boundary?a phospholipid membrane. Yet, association with lipid bilayer is critical for these proteins to function in transport, signal transduction, biosynthesis, and other cellular processes. Membrane proteins that catalyze electron transport are even more difficult to study because they often include multiple chromophores and fluorophores hindering spectroscopic interrogation. In this project, we are developing a powerful application of Nuclear Magnetic Resonance (NMR) and transient absorption (TA) spectroscopies to enable analysis of such chromophore-rich redox systems. Our case study is the cytochrome P450 oxidoreductase (POR) that supplies electrons to the cytochromes P450 localized in endoplasmic reticulum (ER) membrane of liver cells and other tissues. The presence of a phospholipid membrane is strictly required for a productive interaction of P450 with POR, which needs to assume an open conformation to allow for P450 binding. According to literature reports and our own experiments, the isolated soluble cytosolic fragment of POR is auto-inhibited and adopts a predominantly closed state in solution leading to negligible interaction with P450. We hypothesize that membrane binding triggers an open conformation of POR and relieves its auto-inhibition. This open state is further stabilized by binding of the cytochrome P450. We propose to test this hypothesis with the following Specific Aims: (1) Determine the preferential conformational state of oxidized and reduced membrane-bound POR; and (2) Create a structural model of membrane-bound POR in reduced and oxidized states in the presence and absence of cytochrome P450. In Aim 1, we will measure the distances between specific sites in POR using FRET with transient absorption (TA) spectroscopy?a method we recently introduced. We expect to determine the distribution of the donor-acceptor distances in a series of POR samples, which will test our hypothesis that the open-closed equilibrium of membrane-bound POR shifts upon reduction and interaction with P450 to a more open population. In Aim 2, we will establish the relative orientation of the cytosolic domain of POR near the membrane through mapping its contacts with the membrane surface. The membrane contacts will be detected paramagnetic relaxation enhancement (PRE) of methyl-TROSY signals from POR. The membrane contacts information combined with data on the open-closed transition will be used as structural restraints to create a molecular model of POR-nanodisc and POR- nanodisc-P450 complexes which will be further refined with molecular dynamics. This work is significant because we aim to obtain the first structural model of membrane-bound POR to help formulate testable hypotheses on the role of specific mutations in the POR function. The second significant implication is development of the innovative approach based on a combination of the TA and NMR spectroscopies, which will be widely useful for structural analysis of flexible chromophore-rich membrane proteins.